Database Statistics & Information
Feature Description
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Basic statistics and metrics for the AADB database (v1.2).
Distribution of sequences across different pH stress response functional systems.
Distribution of sequences across different microbial species and environments.
AADB groups genes into key functional systems for pH homeostasis and stress adaptation. These interconnected systems protect cells and maintain viability in both acidic and alkaline environments.
1. Glutamate Decarboxylase System (GAD)
The glutamate decarboxylase (GAD) system is one of the most potent acid resistance mechanisms in enteric bacteria like E. coli. It functions by consuming cytosolic protons through the decarboxylation of glutamate to γ-aminobutyric acid (GABA), catalyzed by two pyridoxal-5′-phosphate-dependent isoenzymes, GadA and GadB.
An inner-membrane antiporter (GadC) exchanges the intracellular GABA for external glutamate, effectively exporting one proton per cycle and raising cytosolic pH. Each glutamate decarboxylation cycle exports one proton out of the cell (as part of GABA), thereby enhancing survival in strongly acidic environments (pH ~2–3).
The GAD system is maximally active at moderately low pH (around 4.0–5.5) and is virtually inactive at neutral pH, ensuring it engages only under acid stress. This allows cells to maintain their internal pH above ~5 even when the external pH drops to lethal levels.
Key genetic components include the gadA and gadB genes (encoding the decarboxylases) and gadC (encoding the Glu/GABA antiporter). Expression is tightly regulated by global regulators and two-component systems responsive to acid, such as the transcriptional activator GadE and the EvgS/EvgA system. The GAD system is highly conserved in enteric bacteria and is considered the primary defense during extreme acid challenges.
2. Antiporter System
The antiporter system encompasses membrane transporters that exchange ions (typically cations) for protons across the cell membrane to maintain pH homeostasis. Chief among these are Na+/H+ antiporters, which are ubiquitous in bacteria and help expel sodium in exchange for importing protons.
In E. coli, the major Na+/H+ antiporter NhaA is electrogenic, coupling the inward transport of 2 H+ to the export of 1 Na+. Notably, NhaA activity is strongly pH-dependent: it is almost inactive at acidic pH (below 6) but becomes highly active as the cytoplasm alkalinizes (between pH 6.5–8.5, activity increases ~2000-fold).
This allows E. coli to efficiently remove Na+ and import protons under alkaline stress, preventing internal pH from rising too high. Other antiporters, such as NhaB and ChaA, have overlapping roles to ensure pH stability over a range of conditions.
In extreme alkaliphiles like Bacillus pseudofirmus (which can grow at pH ≥10), multi-subunit antiporters (e.g. the Mrp complex) are crucial for survival. These bacteria maintain a cytoplasmic pH that is 2–2.3 units lower than the external pH, primarily by continuously expelling Na+ while symporting or antiporting H+ into the cell. The antiporter systems generally function across a broad pH range, protecting cells from mild acid stress and being indispensable under alkaline conditions.
3. Acid-Activated Chaperone System
Extremely low pH not only inundates cells with protons but can also denature periplasmic proteins. Enteric bacteria have evolved acid-activated chaperones, notably HdeA and HdeB, to protect periplasmic proteins from aggregation under acid stress.
HdeA and HdeB are small chaperones that are unusual in that they are inactive when folded at neutral pH but become activated by acid-induced structural changes. HdeA functions as a "conditionally disordered" chaperone: at pH > 3.5 it exists as a folded dimer with no client binding activity, but upon exposure to pH ~2–3 it undergoes partial unfolding and dissociates into monomers.
This unfolding exposes hydrophobic surfaces that allow HdeA to bind and stabilize unfolded periplasmic proteins, preventing their aggregation in the acidified periplasm. HdeA's chaperone activity is optimal around pH 2, and upon return to neutral pH it gradually releases client proteins for refolding and reverts to its inactive dimeric state.
HdeB, a paralog of HdeA, plays a complementary role. It remains more structured at low pH and has a higher optimal activation range (pH 4–5), functioning best under moderately acidic conditions where HdeA is less effective. Both HdeA and HdeB are widely conserved in acid-tolerant enteric bacteria and allow bacteria to maintain periplasmic enzyme function and integrity even when external pH is at or below 3, such as in the stomach.
4. Alkaline Resistance System
Bacteria exposed to high pH (alkaline conditions) face the challenge of maintaining sufficient proton availability for cellular processes. Alkaline resistance systems are a collection of strategies that cells use to acidify their cytoplasm and guard against hydroxide ion stress when external pH is high.
A central component is the use of Na+/H+ antiporters to import protons. For instance, E. coli strongly activates the NhaA antiporter at pH > 8, enabling the cell to bring in protons in exchange for expelling Na+. Concurrently, E. coli will down-regulate proton-consuming processes in the respiratory chain and up-regulate alternatives that consume fewer protons.
Adaptation to high pH often involves modifications to the cell envelope as well. Alkaliphilic Bacillus species fortify their cell walls with acidic polymers and alter membrane lipid composition to reduce proton leakage and facilitate proton retention at the membrane surface.
These extremophiles typically rely on a sodium-motive force in addition to proton-motive force. Crucially, alkaliphiles maintain an inwardly directed ΔpH. For example, B. pseudofirmus OF4 growing at external pH 10.5 keeps its cytoplasm around pH 8.2 (over 2 pH units lower). This is achieved by active proton uptake through the Mrp antiporter system and by tightly tethering proton uptake to sites of consumption before protons can diffuse away. Overall, the alkaline resistance system ensures cells can sustain a cytoplasmic pH near neutrality even when living in environments up to pH 10–11.
5. pH Response Regulatory System
Bacteria sense and respond to pH changes through dedicated regulatory circuits that adjust gene expression of pH-homeostasis systems. A key regulatory hub is the two-component regulatory systems that detect external pH or its effects and trigger adaptive responses.
In E. coli, for example, the EvgS/EvgA two-component system responds to acid stress by activating a cascade that includes transcriptional regulators like YdeO and GadE, ultimately inducing the glutamate decarboxylase and other acid resistance genes. Similarly, the ArcB/ArcA system can modulate acid resistance genes under fermentative conditions, and the PhoQ/PhoP system is activated in mildly acidic, low Mg2+ environments.
One well-characterized pH-specific regulator in E. coli is CadC, part of the lysine decarboxylase (Cad) system. CadC is an inner-membrane transcriptional activator that senses acidic pH (around 5.5) in the presence of lysine and induces the cadBA operon. It essentially functions as a pH sensor: at low pH, protonation events enable CadC to activate gene expression, whereas at higher pH CadC is inactive.
Crucially, the RpoS (σS) sigma factor governs a general stress regulon that includes many acid resistance functions. Multiple small RNAs (e.g. DsrA, RprA, ArcZ) increase RpoS translation in response to low pH and other stresses. In summary, the pH response regulatory system is an intricate network of two-component sensors, dedicated pH-responsive transcription factors, and global regulators that together ensure the appropriate suite of genes is turned on to counteract either acid or alkali stress.
6. General Stress Response System
Acid and alkali stresses trigger broader cellular stress responses beyond pH-specific mechanisms. The general stress response, largely orchestrated by the σS (RpoS) regulon in Gram-negatives, is a pivotal system that boosts survival under a variety of hostile conditions including extreme pH.
RpoS controls genes for oxidative stress defense, osmoprotection, and DNA repair, many of which also contribute to acid tolerance. For instance, acid exposure can damage DNA and proteins: E. coli counters this via RpoS-induced DNA repair systems (e.g. the SOS response and nucleotide excision repair) and by upregulating molecular chaperones and proteases to refold or remove damaged proteins.
Under acid stress, cells increase production of chaperones such as GroEL/GroES and DnaK/DnaJ, and proteases like ClpP, to handle misfolded cytoplasmic proteins, complementing the periplasmic HdeA/B system. They also activate DNA repair enzymes (RecA, UvrAB, etc.) to fix acid-induced DNA lesions, ensuring genomic integrity.
Another aspect is cross-protection: exposure to one stress (e.g. mild acid) can induce general stress proteins that incidentally protect against other stresses. The acid tolerance response (ATR) observed in some bacteria during gradual acidification involves transient expression of stress proteins that help cells survive a subsequent pH drop. In summary, the stress response system provides a supportive safety net that works alongside dedicated pH-specific systems, globally protecting cellular components and increasing robustness.
7. Periplasmic pH Regulation System
Gram-negative bacteria possess a periplasmic space between their inner and outer membranes, and under acid stress this compartment can rapidly equilibrate with the acidic environment. Some pathogens have evolved strategies to specifically modulate periplasmic pH as a buffer zone.
The classic example is Helicobacter pylori, which uses a periplasmic buffering system to survive the gastric pH of ~1–2. H. pylori expresses a highly active urease enzyme in the cytoplasm and associates it near the inner membrane. When external pH drops below ~6, a pH-gated urea channel (UreI) in the inner membrane opens to influx urea from the stomach into the bacterial cell.
Cytoplasmic urease then hydrolyzes urea into carbon dioxide (CO2) and ammonia (NH3). These products diffuse or are transported into the periplasm, where CO2 is rapidly converted to bicarbonate (HCO3–) by a periplasmic carbonic anhydrase. The ammonia, being a weak base, captures free protons to form NH4+, while bicarbonate buffers additional H+.
Through this process, H. pylori maintains its periplasm around pH 6.0–6.2 even when the external environment is far more acidic. This localized pH elevation (essentially a "cloud" of ammonia and bicarbonate) protects periplasmic enzymes and allows the bacterium to continue functioning in the stomach. Beyond H. pylori, other bacteria have simpler periplasmic pH defenses using amino-acid-based systems that export basic amines into the periplasm.
8. Membrane pH Transport System
The membrane pH transport system refers to primary proton pumps and other transporters embedded in the cytoplasmic membrane that directly move protons (or hydroxide ions) to control intracellular pH. A prime example is the F1F0-ATPase (ATP synthase).
This enzyme normally uses the proton gradient to generate ATP, but under severe acid stress it can reverse operation: by hydrolyzing ATP, it actively expels H+ from the cytoplasm to the periplasm. This ATP-driven proton pump helps consume excess internal protons when the cell is experiencing a rapid pH drop. E. coli's F1F0-ATPase is known to be upregulated in acid conditions and is quickly engaged to prevent lethal cytosolic acidification.
Electron transport chain components also contribute to membrane pH homeostasis. The NADH dehydrogenase I (NDH-1) in many bacteria pumps protons out of the cytoplasm as it transfers electrons, and its activity can influence cytoplasmic pH. In acid environments, bacteria might down-regulate certain proton-pumping respiratory complexes to avoid adding more H+ to an already acidic cytosol.
Under alkaline stress, Bacillus species use their respiratory chain in conjunction with membrane transporters to generate a proton gradient. Another element is K+ transport, which can help counterbalance charge when H+ is extruded. In summary, the membrane pH transport system provides the energetic means to forcefully adjust proton levels on each side of the membrane, especially during acute pH shocks when passive or secondary transport alone is insufficient.
9. Glutaminase System
The glutaminase-dependent system is a recently identified acid resistance mechanism that parallels the GAD system. Instead of glutamate, this system utilizes L-glutamine as a substrate for acid stress amelioration. The key enzyme is an acid-activated glutaminase (in E. coli, encoded by ybaS, also called GlsA) that deaminates glutamine to glutamate and ammonia (NH3).
At low pH, NH3 readily protonates to NH4+, consuming a proton and thereby raising the pH. Simultaneously, the glutamate produced can feed into the glutamate decarboxylase (GAD) pathway: E. coli's GadC antiporter can import glutamine and export glutamate or GABA, effectively linking the two systems.
When glutamine supply is available, this AR2_Q system couples two proton-consuming reactions: glutaminase yields NH3 (neutralizing H+) and GAD converts glutamate to GABA (exporting H+ out). Overall, the glutaminase system augments acid resistance by providing extra substrate (glutamate) for the GAD pathway and by directly producing ammonia as a cytosolic base.
This system was first delineated in E. coli and shown to markedly improve survival in extreme acid (pH ~2.5) when glutamine is present. The glutaminase system extends the effective pH range of acid resistance: glutamate decarboxylase alone works best to about pH 2.5, but with glutaminase providing extra proton-scavenging, cells gain additional resilience. Homologous acid-active glutaminases have been found in other bacteria such as Lactobacillus reuteri and Brucella species, suggesting the AR2_Q system is widespread.
10. Urease-Mediated Neutralization System
The urease system is a powerful alkalinizing mechanism employed by certain bacteria to survive in strongly acidic environments by producing ammonia. Urease is an enzyme that hydrolyzes urea into ammonia (NH3) and carbon dioxide (CO2). The ammonia then acts to neutralize acid (NH3 + H+ → NH4+), effectively increasing the local pH.
The most famous example is Helicobacter pylori in the stomach: this neutralophilic bacterium relies on urease to withstand gastric acidity and colonize the stomach lining. H. pylori produces urease in abundance (it constitutes up to 10% of total protein) and localizes it in the cytoplasm and just inside the inner membrane.
In an acidic stomach, urea (supplied from gastric fluids and blood) diffuses into the bacterium via the UreI channel, and urease rapidly converts it to NH3 and CO2. The result is a microenvironment around the bacterium that is buffered: ammonia and bicarbonate (from CO2 hydration) raise the pH immediately surrounding the cell, creating a "cloud" that protects H. pylori from pH ~2 acid in the bulk medium.
Expression and activation of urease are tightly regulated. H. pylori has both acute and chronic acid response phases for urease: acutely, the UreI channel opens at low pH to flood substrate in and immediately elevate periplasmic pH; chronically, gene regulatory systems induce higher production of urease when the bacteria experience sustained moderate acidity. The effectiveness of urease is evident: H. pylori can survive for hours at pH 1.0 if urea is available, maintaining its cytoplasmic pH near neutrality and periplasm ~pH 6, a feat unattainable without this enzymatic "acid shield."
11. Other pH-Related Mechanisms
Bacteria possess several additional pH-resistance systems beyond the major ones above, often centered on alternative amino acid catabolism pathways that consume protons or release basic products. In E. coli and many enterics, there are at least three amino-acid decarboxylase systems aside from GAD that function in acid tolerance.
The arginine decarboxylase system (AR3) uses arginine decarboxylase (AdiA) to convert arginine into agmatine, with the consumption of a proton, and an antiporter (AdiC) to export agmatine in exchange for external arginine. This system is induced under anaerobic, acidic conditions and helps survival around pH 4–5. The lysine decarboxylase system (Cad, AR4) consists of lysine decarboxylase (CadA) which produces cadaverine plus CO2, and the CadB antiporter that swaps extracellular lysine for intracellular cadaverine. This system is tuned to mildly acidic environments (optimal around pH 5.5–6).
The ornithine decarboxylase system (SpeF/PotE) converts ornithine to putrescine (another polyamine) plus CO2, consuming H+ in the process. PotE is the corresponding antiporter that exports putrescine in exchange for ornithine. This system has an optimal range in the mid pH 5's and is usually induced during stationary phase or in nutrient-poor acid conditions.
Another important mechanism, especially in Gram-positive bacteria and oral bacteria, is the arginine deiminase (ADI) pathway. This converts arginine to ammonia and carbon dioxide (via citrulline and carbamoyl phosphate intermediates), yielding ATP. The released ammonia neutralizes acid, and the ADI system is unusually acid-tolerant, proceeding even at pH ~3.3 in some lactic acid bacteria. While each of these "other" systems may individually confer only modest pH protection, together they form a network of secondary defenses ensuring continuous coverage across various pH ranges.
Current Version: v1.2
Creation Date: 2025-09-30
Last Updated: 2025-12-23
Version History:
- v1.2 (Current): 728 sequences, aligned sequences, enhanced annotations
- v1.1 (2025-09-30): 907 sequences, 11 functional systems (contains duplicates)
- v1.0 (2025-06-30): 509 sequences, 16 genes
- Multi-environment microbial genome data (acidic and alkaline environments)
- Published pH stress resistance-related research literature
- Functionally verified high-quality sequence data
- Sequence Quality: Strict sequence quality control standards
- Functional Specificity: Functional specificity verification through literature review
- Deduplication: Sequence deduplication processing to ensure uniqueness
- Format Standardization: Consistent FASTA format and annotation standards
- Curated, Non-redundant: Focus on high-confidence, experimentally supported sequences
- pH stress resistance gene identification
- Sequence similarity search
- Functional annotation analysis
- Evolutionary analysis of pH homeostasis mechanisms